We have developed a new way of measuring cytoskeletal actin structure, using Fl-phalloidin to quantitate F-actin organization, and a new way of measuring cytoskeletal function, by the cooperative cellular concentration of a native collagen gel. These two simple, quick in vitro assays can be carried out on cultured precrisis human skin fibroblast-like cells. In our preliminary work, both assays detected significant variation with donor age and culture passage. In addition, we have found that certain inherited diseases of man affect the response of precrisis fibroblasts to these two assays. We now wish to apply these assays, in a prospective scan, to skin fibroblast cells from young individuals with a set of inherited diseases which affect growth control. In order to examine cellular growth requirements for specific hormones, we have been growing mouse fibroblasts in serum-free media with a set of added hormones including Epidermal Growth Factor (EGF) and insulin. Using a novel long term clonal growth assay, we have found that deprivation of insulin causes the greatest growth inhibition of normal cells. Both senescence and transformation affect cellular response in insulin deprivation. We will determine whether cells from normal young individuals need a lesser amount of insulin to divide than do normal cells from aged persons or from young persons with inherited propensity to develop cancer. Insulin binding also will be determined by 125I-insulin in the standard fashion, and also by fluorescein-insulin, both with a low-light intensifier vidicon and with a FACS-IV cell sorter. In many ways, oncogenic transformation and senescence are sets of opposing in vitro changes in cultured fibroblastic cells. The simplest hypothesis linking these opposing sets predicts that the same cellular molecules are involved in both changes. We will probe for this linkage by direct analysis of the effect of inherited cancer, culture passage number or donor age on the capacity of human skin fibroblast populations to respond to the oncogenic virus SV40. The semipermissive interaction of human cells with SV40 DNA makes transformation of these cells difficult, and prevents close analysis of the virus-cell interaction. Origin-minus SV40 DNA cannot replicate in human cells. This DNA, coupled wit (Text Truncated - Exceeds Capacity)